The invention concerns a blood diluent for blood cell counting and sizing and more particularly, a multipurpose blood diluent for use in hematological enumeration of blood cells and the determination of hemoglobin concentration and their collective indices by electronic particle analysis using a Coulter scanning device. Accordingly, the diluent comprises a stable water solution of chemical salts providing an electrolytic solution to which a blood sample can be added so as to dilute the larger number of red blood cells, white blood cells and other blood components and enable the desired parameters of these blood components to be measured, counted and estimated.
It is a common medical diagnostic procedure to analyze and test a blood sample of a patient in order to make certain classic determinations with respect to the blood sample. This procedure is an important tool for the physician. Six characteristically important parameters are referred to as red blood cell count (RBC), the hematrocrit (HCT), the hemoglobin (HGB), the mean corpuscular volume (MCV), the mean corpuscular hemoglobin (MCH) and the mean corpuscular hemoglobin concentration (MCHC). A seventh important determination is white blood cell count (WBC). An instrument which will accept a patient's blood sample and process the sample automatically and continuously to provide the parameters or determinations enumerated is described and claimed in U.S. Pat. No. 3,549,994. Said U.S. Pat. No. 3,549,994 provides acceptable definitions of said parameters and illuminates the problems to be solved in the handling of the blood sample as it is drawn through the fluid system of said patented apparatus.
Coulter Electronics, Inc. of Hialeah, Florida also manufacturers and sells other blood cell counting and analyzing instruments which are less sophisticated than the apparatus of said U.S. Pat. No. 3,549,994 but which are operated to determine red blood cell and white blood cell counts, hemoglobin concentration and their collective indices such as HCT, MCV, MCH and MCHC. The multi-purpose blood diluent embodying the invention is suitable for use with such other instrumentation as well, where problems attendant the successful handling of the blood sample are the same. In other words, this blood diluent is compatible with other electronic particle analysis instruments utilizing the Coulter principle. Such instruments may be referred to herein, at times, collectively by the registered trademark "COULTER COUNTER" owned by Coulter Electronics, Inc.
To contribute to a fuller understanding of the invention, it is helpful to appreciate certain characteristics of red blood cells. Red blood cells are biconcave discs generally toroidal in shape, much like a doughnut. The intermembrane is elastic. Hemoglobin and other components are contained in the interior of the cell. The specific parameters of the red blood cell which it is clinically desirable to measure by the electronic instrumentation using the Coulter principle dictate the necessary characteristics of a suitable diluent.
For instance, it is desirable to know the volume within the red blood cell. Once this measurement is ascertained and the red blood cells have been counted, the packed cell volume or Hematocrit (HCT) can be computed. The diluent of the invention therefore must be an electrolyte which enables electronic measurements to be made by the Coulter type instrument. The diluent of the invention also should be capable of equilibrating and stabilizing the volume of red blood cells in the sample so that its cubic volume can be measured, namely, MCV.
As explained in said U.S. Pat. No. 3,549,994, the enumeration of blood cells by the "Coulter Counter" requires accurate and successful dilution of the blood sample drawn into its fluid system. Such analysis predicates certain diluent specifications essential to the successful and proper performance of the apparatus. For instance, the diluent must be capable of maintaining the chemical and physical integrity of blood corpuscles prior to and during the assay procedure. The blood cells are required to retain the same physical character in the diluted solution as exhibited in the undiluted sample. For this purpose, the blood diluent must be isotonic and osmotically balanced relative the solutions in the blood cells. The resistance of the red blood cells to lysing for purposes of hemoglobin determinations must not be altered in any way by the blood diluent. The blood diluent must not interfere in any way with the process of converting the hemoglobin released to a quantitatively determinative hemoglobin compound, such as, cyanmethemoglobin, so that the accuracy of the hemoglobin determination will not be compromised.
A suitable blood diluent must be devoid of foreign particulate matter because the presence of foreign particles will result in the enumeration thereof as a blood cell or constituent. Thus, the blood diluent must be filtered free of particles exceeding 0.2 micron diameter at the time of manufacture. Concomitantly, the diluent must be bacteriostatic in nature so as to prevent the growth of microorganisms after packaging of the diluent. It has been recognized that a proper blood diluent for use with such electronic particle analysis apparatus and hemoglobinometer instruments must be unreactive and osmotically balanced if reproducible, accurate results are to be obtained.
Such electrolyte solutions used in blood cell counting and sizing are required to be of such concentration that the electrolyte ions exert an osmotic pressure equal to that of the intracellular fluid. If the cells are suspended in a solution of reduced osmotic pressure, i.e., hypotonic, the cells will absorb water and expand until burst thereof releases the cell fluids into the solution. This condition is called "hemolysis". Where blood cells are suspended in an electrolyte solution of increased osmotic pressure, cellular fluid will be lost to the solution thereby shrinking the volume of the cell. This condition is called "crenation". Although preservatives for preventing bacterial or fungal growth are desirable, caution must be observed to avoid increasing the cell volume of blood cells in suspension by reason of the preservative used.